This project seeks to develop new therapies for diabetic retinopathy by targeting the urokinase/urokinase receptor system (uPA/uPAR). Our previous work has shown that diabetes/high glucose-induced injury of the retinal vasculature is mediated by oxidative stress-induced increases in VEGF expression, which causes breakdown of the blood-retinal barrier due to activation of the uPA/uPAR system. Our preliminary data link these events to diabetes' action in decreasing the expression of the anti-angiogenic, neuro-trophic growth factor pigment epithelial derived factor (PEDF). PEDF is known to block the angiogenic and permeability-inducing functions of VEGF. Studies of diabetic retinopathy and diseases characterized by breakdown of the blood-retinal barrier and retinal neovascularization have shown that increases in retinal VEGF are correlated with decreases in PEDF. Oxidative stress reduces PEDF by increasing the formation of matrix metalloproteinases 2 and 9 (MMP2, MMP9), which degrade and inactivate PEDF. Our preliminary data suggest that diabetes-induced increases in uPAR are associated with increases in MMP9 and decreases in PEDF. Moreover deletion of the uPAR gene prevents MMP9 release, preserves PEDF and protects the blood-retinal barrier. We also have data showing that diabetes-induced neuronal/glial cell death is correlated with decreases in PEDF. Based on these data, we hypothesize that diabetes and high glucose induce breakdown of the blood-retinal barrier and neuro-glial cell death by causing activation of the uPA/uPAR system and decreasing PEDF. This hypothesis will be tested by experiments using a combination of cell and molecular biology approaches in specific aims to test the following hypotheses: 1. uPAR is required for diabetes- induced breakdown of the blood-retinal barrier and neual/glial cell death via activation of uPA/uPAR. 2. Inhibiting uPA proteolytic activity will block diabetes-induced decreases in retinal PEDF levels and prevent breakdown of the blood-retinal barrier and neural/glial cell death. 3. Activation of the uPA/uPAR system causes increases in paracellular permeability viauPA- mediated proteolysis. 4. PEDF inhibits VEGF or high glucose-induced increases in paracellular permeability by blocking the uPA/uPAR expression pathway. These studies will set the stage for developing therapies for targeting both neural and vascular pathology and preventing diabetic retinopathy, the leading cause of blindness in working age adults in the US today.